6 research outputs found
Bioimaging with NP-angiography showing GFP expression using the Topcon camera with fluorescein angiography filter settings.
<p>Late phase FAs (A and D) show the CNV lesions prior to injection of NP.
Autofluorescent images taken prior to injection of NP reveal minimal
background fluorescence of the CNV lesions (B and E). Injection of
targeted NP carrying a GFP plasmid (NP-GFPg) causes increased
fluorescence of the CNV lesions from GFP expression (C) whereas
non-targeted NP carrying a GFP plasmid (ntNP-GFPg) does not cause any
increase in the intensity of fluorescence of the CNV over background
autofluorescence (F).</p
Choroidal flatmounts showing accumulation of rhodamine labeled NP and expression of GFP plasmid in the CNV.
<p>The CNV lesions are delineated by arrowheads in bright field images with
false blue color (A and E). FITC-filtered images highlight the GFP
expression one day after systemic injection of Rd-NP-GFPg (B) whereas
non-targeted NP (Rd-ntNP-GFPg) does not induce GFP expression in CNV
(F). Cy3-filtered images highlight that rhodamine-labeled NP
(Rd-NP-GFPg) accumulates in the CNV (C), while rhodamine-labeled
non-targeted NP (Rd-ntNP-GFPg) does not (G). Some particles can be
visualized circulating in the choroidal vessels. Overlay of images
A–C is presented in panel D and overlay of E–G is shown in
H.</p
Late phase fluorescein angiography (FA) and choroidal flatmounts (<i>x10</i>) two weeks after laser photocoagulation.
<p>Representative lesions are from the control group (A–D) and the
NP-ATPμ-Raf treated group (E and F). Group (A) received no
treatment; (B) received intravenous injection of non-targeted NP
containing ATPμ-Raf on days 1, 3, and 5 after laser CNV creation;
(C) received intravenous injection of
α<sub>ν</sub>β<sub>3</sub> targeted-NP without
ATPμ-Raf gene on days 1,3, and 5; (D) received injection of
ATPμ-Raf gene without NP on days 1, 3, and 5; (E) received injection
of α<sub>ν</sub>β<sub>3</sub> targeted-NP containing
ATPμ-Raf (NP-ATPμ-Raf) on days 1, 3, and 5; and (F) received
injection of NP-ATPμ-Raf on days 3, 5, and 7. NP-ATPμ-Raf
treated groups (E and F) had significantly lower grade CNV lesions on FA
grading and smaller CNV size compared to the control group (A–D).
No statistically significant difference in size was noted between the
control groups A–D. Quantification of the CNV size on choroidal
flat mounts is shown in (G). *P<0.01. Data are expressed as the
mean ± SE.</p
Increased macrophage infiltration at the site of treated CNV.
<p>Macrophage infiltration was highest on day 3 with gradual decrease on
days 5 and 7. Significantly higher number of macrophages were observed
with the NP-ATPμ-Raf treated group compared to the control group on
days 5 and 7 (A and B). There was a statistically significant reduction
of CNV size noted on day 7(C). Immunofluorescent staining of
representative frozen sections (<i>x20</i>) obtained at 3, 5,
and 7 days after laser photocoagulation for ED 1, a marker for
macrophage (D). *P<0.01. Data are expressed as the mean ±
SE.</p
Evaluation of endothelial cell apoptosis with TUNEL staining in frozen sections.
<p>Quantification of TUNEL positive cells showed significantly more
TUNEL(+) cells/lesion (A) and TUNEL (+) cells/mm<sup>2</sup>
(B) with treatment of NP-ATPμ-Raf compared to the control group on
day 3 and 5 after laser injury. There was a statistically significant
reduction of CNV size noted on day 7(C). Double-immunofluorescent
staining of frozen sections (<i>x20</i>) obtained at 3, 5 and
7 days after laser photocoagulation for the endothelial cell marker CD31
and TUNEL stain (D). *P<0.01. Data are expressed as the mean
± SE.</p
<i>In vivo</i> evaluation of CNV utilizing SD-OCT.
<p>Quantification of CNV size using SD-OCT (A) reveals a decrease in CNV
size, reaching statistical significance on day 7 (Mann- Whitney U test,
p = 0.001) in the NP-ATPμ-Raf treated group
compared to the control group. A hyper-reflective subretinal lesion is
seen as delineated by the red dotted line (B). This lesion corresponds
to the hyporeflective area on fundus reconstruction (red dotted circle,
C). *P<0.01. Data are expressed as the mean ± SE.</p